Methods and compositions for reducing bacterial tolerance to antibacterials, disinfectants and organic solvents

ABSTRACT

The invention relates to methods and compositions for manipulating bacterial resistance to non-antibiotic antibacterial compositions, disinfectants and organic solvents. The invention provides methods for rendering bacterial cells susceptible to non-antibiotic antibacterial compositions. Also provided are methods to reduce the selection of bacterial mutants having an multiple antibiotic resistance phenotype by non-antibiotic antibacterial compositions. The invention also provides methods for testing the ability of non-antibiotic antibacterial compositions to select for or induce a multiple antibiotic resistance phenotype in bacteria. Also provided are methods for increasing or decreasing bacterial tolerance to organic solvents by increasing or decreasing the activity of bacterial organic solvent efflux pumps. Compositions useful in the foregoing methods are also provided.

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from PCTapplication serial number unknown (attorney's docket numberT0359/7007WO), filed Oct. 2, 1997, and U.S. provisional applicationserial No. unknown (attorney's docket number T0359/7007), filed Oct. 3,1997.

GOVERNMENT SUPPORT

[0002] This work was supported in part by U.S. Public Health Servicegrant number GM51661. The government may have certain rights in thisinvention.

FIELD OF THE INVENTION

[0003] This invention relates to methods and compositions formanipulating bacterial resistance to non-antibiotic antibacterialcompositions, disinfectants and organic solvents.

BACKGROUND OF THE INVENTION

[0004] Antibiotic or antimicrobial substances have long been used toinhibit the growth of bacteria or other microbes and to treat bacterialor microbial infections in humans, other animals, and in tissue culture.The use of antibiotics or antimicrobials in a treatment regimen,however, has the undesirable effect of selecting for bacteria or othermicrobes which are resistant to those antibiotics or antimicrobialswhich are administered or applied. As a result, treatment regimens canbe adversely affected or, in some cases, rendered ineffective. Thisnecessitates a continual search for new antibiotics and antimicrobials.

[0005] Of particular interest is the discovery of bacteria which expressa multiple antibiotic resistance phenotype (Mar). This phenotype entailssimultaneous resistance to a multiplicity of antibiotics which areunrelated in chemical structure. The appearance of such bacteria andinfections by such bacteria greatly increase the difficulty ofidentifying effective antibiotics and treating infections in humans orother animals.

[0006] Multiple antibiotic resistance in bacteria is most commonlyassociated with the presence of plasmids and/or transposons whichcontain one or more resistance genes, each encoding a single antibioticresistance phenotype. Multiple antibiotic resistance associated with thechromosome, however, has been reported in Klebsiella, Enterobacter,Serratia (Gutmann et al., J. Infect. Dis. 151:501-507, 1985), Neisseria(Johnson and Morse, Sex. Transm. Dis. 15:217-224, 1988), and Escherichia(George and Levy, J. Bacteriol. 155:531-540, 1983).

[0007] Bacteria expressing a chromosomal multiple antibiotic resistancephenotype can be isolated by selecting bacteria with a single antibioticand then screening for cross-resistance to structurally unrelatedantibiotics. For example, George and Levy initially described achromosomal multiple antibiotic resistance system which exists inEscherichia coli and which can be selected by a single drug, e.g.,tetracycline or chloramphenicol (George and Levy, 1983). In addition toresistance to the selective agents, the Mar phenotype includesresistance to structurally unrelated agents, including nalidixic acid,rifampin, penicillins, and cephalosporins (George and Levy 1983) as wellas fluoroquinolones (Cohen et al. 1989).

[0008] The chromosomal gene locus which correlates with the Marphenotype observed in E. coli has been identified. The chromosomal marlocus, located at 34 min on the E. coli chromosomal map, is involved inthe regulation of intrinsic susceptibility to structurally unrelatedantibiotics (Cohen et al., J. Bacteriol. 175:1484-1492, 1993; Cohen etal., Antimicrob. Agents and Chemother. 33:1318-1325, 1989; Cohen et al.,J. Bacteriol. 170:5416-22, 1988; Goldman et al., Antimicrob. AgentsChemother. 40:1266-1269, 1996), as well as the expression of antioxidantgenes (Ariza et al., J. Bacteriol. 176:143-148, 1994; Greenberg et al.,J. Bacteriol. 173:4433-4439, 1991) and internal pH homeostasis (Rosnerand Slonczewski, J. Bacteriol. 170:5416-22, 1994). The mar locusconsists of two transcription units (marC and marRAB) which aredivergently transcribed from a central putative operator-promotor region(marO) (Cohen et al., 1993; Goldman et al., 1996). marR is the repressorof the marRAB operon (Cohen et al., 1993; Martin and Rosner, Proc. Natl.Acad. Sci. USA 92:5465-5460, 1995; Seoane and Levy, J. Bacteriol.177:3414-3419, 1995). Mutations in marR result in increased expressionof the marRAB operon. Overexpression of marA alone is sufficient toproduce the multiple antibiotic resistance phenotype (Cohen et al.,1993; Gambino et al., J. Bacteriol. 175:2888-2894, 1993; Yan et al.,Abstr. A-26, p. 5, In Abstracts of the 1992 General Meeting of theAmerican Society for Microbiology, American Society for Microbiology,Washington, D.C., 1992). marB has no effect of its own; however, when itis present on the same construct with marA, it produces a small increasein antibiotic resistance (White et al., Abst A-104, p. 20. In Abstractsof the 1994 General Meeting of the American Society for Microbiology,American Society for Microbiology, Washington, D.C. 1994). The functionof marC is unknown; however, it also appears to enhance the multipleantibiotic resistance phenotype when cloned on the same DNA fragmentwith the marRAB operon (Goldman et al., 1996; White et al., 1994).

[0009] Overexpression of marA confers multiple antibiotic resistance viaincreased efflux of antibiotics, including fluoroquinolones,tetracycline, and chloramphenicol (Cohen et al., 1989; George and Levy,1983; McMurry et al., Antimicrob. Agents Chemother. 38:542-546, 1994).Transcription of the acrAB operon, which encodes a multi-drug effluxpump whose expression is modulated by global stress signals (Ma et al.,Mol. Microbiol. 16:45-55, 1995; Ma et al., Mol. Microbiol. 19:101-112,1996), was shown to be elevated in strains containing marR mutations anddisplaying the Mar phenotype (Okusu et al., J. Bacteriol. 178:306-308,1996). Moreover inactivation of acrAB led to increased antibioticsusceptibility in wild type and Mar mutants (Okusu et al., 1996).

[0010] More recently, mutations of marR have been found in clinicalisolates resistant to quinolones (Maneewannakul and Levy, 1996). Thusmar mutants can be selected under clinical conditions and not merelyunder controlled laboratory conditions. Early mar mutants (i.e.,“first-step” mar mutants) remain susceptible to many common antibiotics,although such mutants can achieve levels of clinical resistance tocertain antibiotics, including tetracycline, nalidixic acid and rifampin(reviewed by Alekshun and Levy, Antimicrob. Agents Chemother.41:2067-2075, 1997). First-step mar mutants thus may serve as precursorsof bacterial mutants which display higher levels of resistance resultingfrom additional mutations on the chromosome. Thus it has beendemonstrated that antibiotics can select for mutations in chromosomalgene loci which confer multiple antibiotic resistance under clinicalconditions.

[0011] Non-antibiotic antibacterial compositions such as disinfectantsare widely used in both clinical and consumer environments for reducingbacterial contamination of work surfaces, equipment, products and thelike. These non-antibiotic antibacterial compositions have beenincorporated into a wide spectrum of cleansers, disinfectantcompositions, soaps, lotions, plastics, etc. It is not known whetherexposure of bacteria to non-antibiotic antibacterial compositions alsocan select for bacterial mutants, including those which display amultiple antibiotic resistance phenotype.

SUMMARY OF THE INVENTION

[0012] It has now been discovered that bacterial mutants having multipleantibiotic resistance can be selected by non-antibiotic antibacterialagents such as common disinfectants. It further has been discovered thatthe phenotype of the multiple antibiotic resistant mutants selected by anon-antibiotic antibacterial agent results from mutations in chromosomalgene loci which regulate expression of efflux pumps, which loci havebeen implicated in multiple antibiotic resistance phenotypes asdescribed above. The efflux pumps actively pump out the non-antibioticantibacterial agents, as well as organic solvents and antibiotics,thereby rendering the mutant bacteria resistant to all of the foregoingcompounds.

[0013] According to one aspect of the invention, a method is providedfor inhibiting the selection and/or propagation of a bacterial mutantthat overexpresses an efflux pump. Bacteria are contacted with an agentthat binds to a gene locus (the expression of the gene locus enhancesexpression of the efflux pump) or an expression product thereof, in anamount effective to inhibit the gene locus-enhanced expression of theefflux pump. In preferred embodiments, the gene locus is selected fromthe group consisting of a mar locus, a sox locus and a rob locus. Alsoin preferred embodiments, the efflux pump is acr-like, including theacrAB efflux pump.

[0014] The agent can be selected from the group consisting of chemicals,antisense nucleic acids, antibodies, ribozymes, and proteins whichrepress expression of the gene locus. A preferred embodiment is an agentthat is an antisense nucleic acid, and in particularly preferredembodiments, the agent is antisense to the mar locus, sox locus and/orrob locus. Another preferred embodiment is chemical inhibitors of effluxpumps, particularly L-phenylalanyl-L-arginyl-β-naphthylamide.

[0015] According to another aspect of the invention, a method isprovided for rendering bacterial cells more susceptible to anon-antibiotic bactericidal or bacteriostatic agent that is a substrateof an efflux pump. An inhibitor of a gene locus or an expression productthereof is administered to a bacterial cell, wherein the expression ofthe gene locus enhances expression of an efflux pump. In preferredembodiments the gene locus is selected from the group consisting of amar locus, a sox locus and a rob locus. In other preferred embodimentsthe efflux pump is acr-like and can be acrAB. The preferred inhibitorsare as described above.

[0016] According to still another aspect of the invention, a method isprovided for rendering bacterial cells more susceptible to anon-antibiotic bactericidal or bacteriostatic agent that is a substrateof an efflux pump. The method involves administering to the bacterialcell an inhibitor of the efflux pump. In preferred embodiments theefflux pump is acr-like and can be acrAB. Preferably the inhibitor isselected from the group consisting of about 4% ethanol, methanol,hexane, minocycline and L-phenylalanyl-L-arginyl-β-naphthylamide.

[0017] According to another aspect of the invention, a method isprovided for modulating (increasing or decreasing) the ability ofbacterial cells to survive in an organic solvent. In certain embodimentsthe method involves enhancing expression in the bacterial cells of anorganic solvent bacterial efflux pump by growing the bacterial cells inthe presence of a non-mar/sox/rob inducing agent, wherein the agentinduces the overexpression of the organic solvent bacterial efflux pump.The agent can be a gene encoding an acr-like pump, the acrAB pump, orexpression products thereof. In other embodiments the method involvesreducing expression in the bacterial cells of an organic solventbacterial efflux pump by growing the bacterial cells in the presence ofan agent, wherein the agent reduces the expression of the organicsolvent bacterial efflux pump. The agent can be an antisense nucleicacid which binds to a gene locus encoding an acr-like pump, especiallythe acrAB pump, a gene locus which enhances expression of an effluxpump, such as marA, soxA and robA, and the like. The agent also can be aribozyme or a protein which represses expression of the gene locus. Theagent also can be an antibody to an expression product of the foregoinggenes. The agent also can be a chemical compound which reducesexpression of the efflux pump, or reduces activity of the efflux pump,such as L-phenylalanyl-L-arginyl-β-naphthylamide.

[0018] According to another aspect of the invention, a method isprovided for testing the ability of a non-antibiotic composition toinduce a multiple antibiotic resistance phenotype in a bacterium. Thebacterium is contacted with the non-antibiotic composition. Theexpression of a bacterial gene locus is determined, the alteredexpression of which is indicative of induction of the multipleantibiotic resistance phenotype in the bacterium. Then, the result ofthis determination is compared with a control, wherein alteredexpression of the bacterial gene locus indicates that the non-antibioticcomposition induces the multiple antibiotic resistance phenotype in thebacterium. In preferred embodiments, the gene locus is selected from thegroup consisting of a mar locus, a sox locus, a rob locus and anacr-like efflux pump locus. In one particular embodiment the efflux pumplocus is acrAB. The foregoing methods can be carried out using anon-antibiotic composition that is an inactive ingredient. The inactiveingredient can be a non-bactericidal ingredient. The inactive ingredientalso can be a non-bacteriostatic ingredient. In one preferred embodimentthe method is carried out by determining the enzymatic activity of anexpression product of a marker gene, preferably lacZ, fused to thebacterial gene locus.

[0019] According to another aspect of the invention, a composition isprovided. The composition includes a non-antibiotic bactericidal orbacteriostatic first agent and a second agent that inhibits theexpression of activity of an efflux pump. In one embodiment, the secondagent inhibits the expression of a gene locus or an expression productthereof, wherein the expression of the gene locus enhances expression ofthe efflux pump. In preferred embodiments, the second agent is selectedfrom the group consisting of antisense nucleic acids, antibodies,ribozymes and proteins that repress expression of the gene locus. In onepreferred embodiment the second agent inhibits an acr-like efflux pump,and particularly preferred is an antisense nucleic acid. The secondagent also can be selected from the group consisting of 4% ethanol,methanol, hexane, minocycline andL-phenylalanyl-L-arginyl-β-naphthylamide. The preferred second agent isL-phenylalanyl-L-arginyl-β-naphthylamide. The first agent in someembodiments is selected from the group consisting of triclosan, pineoil, quaternary amine compounds including alkyl dimethyl benzyl ammoniumchloride, chloroxylenol, triclocarbon, disinfectants and organicsolvents.

[0020] According to still another aspect of the invention, a method foridentifying an antibacterial composition which does not select for orinduce a multiple antibiotic resistance phenotype in a bacterium isprovided. The bacterium is contacted with the antibacterial composition.The expression of a bacterial gene locus is determined, the alteredexpression of which is indicative of induction of the multipleantibiotic resistance phenotype in the bacterium. Then, the result ofthis determination is compared with a control, wherein alteredexpression of the bacterial gene locus indicates that the antibacterialcomposition induces the multiple antibiotic resistance phenotype in thebacterium and a lack of altered expression of the bacterial gene locusindicates that the antibacterial composition does not induce themultiple antibiotic resistance phenotype in the bacterium. In preferredembodiments, the gene locus is selected from the group consisting of amar locus, a sox locus, a rob locus and an acr-like efflux pump locus.In one particular embodiment the efflux pump locus is acrAB. In onepreferred embodiment the method is carried out by determining theenzymatic activity of an expression product of a marker gene, preferablylacZ, fused to the bacterial gene locus.

[0021] The invention also provides methods for identifyingantibacterials which are not subject to efflux pumps, e.g. thoseantibacterials which are not substrates for efflux pumps. Theseantibacterials are those which have bactericidal or bacteriostaticaction against bacteria which express an efflux pump, particularly thosewhich overexpress an efflux pump, particularly an acr-like pump.especially acrAB. These and other aspects of the invention are describedin greater detail below.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 shows the Northern blot analysis of marRAB mRNA inbacterial mutants.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention is based on the discovery that non-antibioticantibacterial compositions and organic solvents select for mutantbacteria which are resistant not only to the non-antibioticantibacterial compositions, but also to a range of antibiotics (i.e. amultiple antibiotic resistant phenotype) and also to organic solvents.All of the foregoing compounds are pumped out of bacteria by effluxpumps, i.e., the foregoing compounds are substrates for the effluxpumps. Based on these discoveries it is now possible to enhance theantibacterial properties of non-antibiotic antibacterial compositionsand also reduce the selection of bacterial mutants having a multipleantibiotic resistance phenotype by such compositions. The invention alsoprovides methods for testing the ability of non-antibiotic antibacterialcompositions to select for or induce a multiple antibiotic resistancephenotype in bacteria. The invention also provides methods forincreasing or decreasing bacterial tolerance to organic solvents byincreasing or decreasing the activity of bacterial organic solventefflux pumps, such as by increasing or decreasing expression of anefflux pump, increasing or decreasing expression of genes whichpositively regulate efflux pump gene loci, and the like. The inventionfurther provides methods for identifying antibacterial compositionswhich do not select for or induce a multiple antibiotic resistancephenotype in bacteria, such as those antibacterials which are notsubstrates for efflux pumps. Compositions useful in the foregoingmethods are also provided.

[0024] As used herein, a non-antibiotic antibacterial composition is amolecule or combination of molecules which are bactericidal orbacteriostatic, but which are not antibiotics. “Antibiotics” are thosebactericidal or bacteriostatic compounds which are administered in vivoto people, animals or plants which have a bacterial infection, or whichare used in vitro for research on bacterial infections of animals. Anon-antibiotic antibacterial composition is not administered to asubject, but rather is used as a disinfectant for killing bacteria orreducing the growth rate of a population of bacteria. Non-antibioticantibacterial compositions are added as the active ingredients in avariety of industrial and household disinfectants, such as LYSOL™,PINESOL™, and the like. Non-antibiotic antibacterial compositions alsoare added as the antibacterial active ingredient in non-disinfectantcompositions such as soaps, lotions, cleansers and the like. Morerecently, non-antibiotic antibacterial composition have beenincorporated into plastics for making a variety of articles ofmanufacture which have resistance to bacterial growth.

[0025] The non-antibiotic antibacterial compositions, as used herein,may have active and inactive ingredients. The active ingredients are, ofcourse, the bactericidal or bacteriostatic agents which have the effectof slowing or stopping growth of populations of bacteria, or evenkilling such populations of bacteria. Active bactericidal orbacteriostatic ingredients include triclosan, pine oil, quaternary aminecompounds such as alkyl dimethyl benzyl ammonium chloride,chloroxylenol, triclocarbon, and other well known disinfectants. Theinactive ingredients are the balance of the components of thenon-antibiotic antibacterial compositions, including surfactants andother cleansing agents, binders, bulking agents and other compounds.Thus non-antibiotic antibacterial compositions refers both to the activeingredient of the compositions as well as the compositions themselves.

[0026] The invention provides methods for inhibiting the selection orpropagation of a bacterial mutant that overexpresses an efflux pump. By“inhibiting the selection or propagation”, it is meant that the methodprovides inhibition of selection of a multiple antibiotic resistantbacterial mutant (i.e., the initial mutation event which causes theinduction of an efflux pump) and/or inhibition of propagation of amultiple antibiotic resistant bacterial mutant (i.e., growth and/orreplication of such bacteria).

[0027] The invention also provides methods for rendering bacterial cellsmore susceptible to non-antibiotic antibacterial compositions byadministering to the bacterial cells inhibitors of an efflux pump or agene locus which enhances expression of the efflux pump, or anexpression product thereof. By “administered to”, it is meant that thebacterial cells are contacted with the inhibitor for a time sufficientto permit inhibition of the efflux pump or gene locus.

[0028] The invention further provides methods for increasing ordecreasing organic solvent tolerance of bacterial cells. In thesemethods, overexpression of an organic solvent efflux pump is induced ordecreased by growing the cells in the presence of an agent. By induced“overexpression” it is meant that the organic solvent efflux pump isexpressed at a higher level in bacterial cells grown in the presence ofan inducing agent than in identical bacterial cells grown underidentical conditions but without the agent, i.e., a level of expressionthat is sufficient to increase organic solvent tolerance. By reduced“expression” it is meant that the organic solvent efflux pump isexpressed at a lower level in bacterial cells grown in the presence ofan inhibiting agent than in identical bacterial cells grown underidentical conditions but without the agent, i.e., a level of expressionthat is sufficient to reduce tolerance or increase organic solventsusceptibility. These methods can also confer organic solvent toleranceor susceptibility by modulating the activity of an efflux pump asdescribed herein. Organic solvent tolerance or susceptibility can bedetermined by standard methodologies, including those exemplified inExample 2 below.

[0029] One of the features of antibacterial products is the reduction inbacterial populations in those products or on those products, or onsurfaces to which such products are applied. As disclosed herein,non-antibiotic antibacterial products also can select for multipleantibiotic resistant bacteria. It would be useful to be able todetermine which non-antibiotic antibacterial compositions select fordeleterious mutants. Having determined that non-antibiotic antibacterialcompositions can select for mutants, it is also possible that othernon-antibiotic compositions can select for mutations. Therefore theinvention embraces methods for testing the ability of non-antibioticcompositions to induce a multiple antibiotic resistance phenotype. Thesemethods permit testing of any non-antibiotic composition, including theinactive ingredients in cleansers, soaps, disinfectants and the like. Inthese methods, a bacterial culture is contacted with a non-antibioticcomposition and the expression of a gene locus which is indicative of amultiple antibiotic resistant phenotype is determined. The gene locusexpression can be determined by any convenient method, of which many areknown in the art. These methods include enzyme assays comprising fusionsof regulatory loci to a marker gene (e.g. as described for a marregulatory locus in PCT published application WO94/05810), amplificationof gene transcripts (such as using polymerase chain reaction),hybridization methods including Northern blots, and measurement ofprotein expression including Western blots, ELISA, etc. The level ofexpression of the gene locus is then compared with a control todetermine if the non-antibiotic compositions induced the multipleantibiotic resistant phenotype.

[0030] According to the invention, various agents which inhibit theexpression or activity of an efflux pump or gene loci which controlexpression of the efflux pump are useful for reducing selection and/orpropagation of mutant bacteria, and also render the cells moresusceptible to non-antibiotic antibacterial compositions. Theseinhibitors are contacted with or administered to the bacterial cells toprevent the undesirable effects of the non-antibiotic antibacterialcompositions. One convenient way to ensure contact of the appropriatebacterial cell populations is to include the inhibitors and agents inthe non-antibiotic antibacterial compositions. Thus the inventionfurther provides compositions comprising a non-antibiotic bactericidalor bacteriostatic first agent and a second agent which inhibits theexpression or activity of an efflux pump, as described above. Thesecompositions can be prepared according to the standard procedures usedto prepare non-antibiotic antibacterial compositions. For example, astandard disinfectant composition such as PINE-SOL™ can have added to itan effective amount of an inhibitor of an efflux pump such as describedin PCT published patent application WO96/33285, or an antisense nucleicacid which binds to the efflux pump gene locus, etc.

[0031] By “effective amount” is meant an amount of the second agentwhich reduces the selection of mutants by the non-antibiotic firstagent. Effective amounts can be determined using standard bacterialgrowth and mutation assays, including those provided herein. Forexample, various amounts of the second agent can be added to anon-antibiotic antibacterial composition, and the combined compositioncan be used as provided in the examples below to select bacterialmutants. Any amount of the second agent which reduces the number ofmutants selected relative to the number of mutants selected by thenon-antibiotic antibacterial composition alone is an effective amount.One of ordinary skill in the art can determine with no more than routineexperimentation what constitutes an effective amount of a second agent,and what amount of a second agent is optimal to prevent selection ofmutants by the non-antibiotic antibacterial compositions. Effectiveamounts of other inhibitors and agents disclosed herein can bedetermined similarly.

[0032] As disclosed herein, inhibitors of the marA gene locus and otherloci which regulate efflux pumps are effective to reduce the selectionof antibiotic resistant bacterial mutants by non-antibioticantibacterial compositions, and also potentiate the antibacterialproperties of such compositions. The marA gene has been cloned andsequenced, the sequence deposited as GenBank accession number M96235.The marA gene has homologs in E. coli, as well as in other species ofbacteria. Inhibitors of such marA homologs also are useful for reducingthe selection of antibiotic resistant bacterial mutants and potentiatingthe antibacterial properties of non-antibiotic antibacterialcompositions.

[0033] For example, the MarA protein is homologous to both SoxS, theeffector of the soxRS regulon (Fawcett and Wolf, Mol. Microbiol.14:669-679, 1994; Li and Demple, J. Biol. Chem. 269:18371-18377, 1994),and RobA, a small protein that binds to the E. coli replication originand some stress gene promoters (Ariza et al., 1995; Cohen et al., 1995;Jair et al., J. Bacteriol. 178:2507-2513, 1996; Skarstad et al., J.Biol. Chem. 268:5365-5370, 1993). The soxRS regulon mediates the cell'sresponse to oxidative stress (Amabile-Cuevas and Demple, Nucleic AcidsRes. 19:4479-4484, 1991; Nunoshiba et al., J. Bacteriol. 174:6054-6060,1992; Wu and Weiss, J. Bacteriol. 173:2864-2871, 1991). soxS genesinclude those found in S. typhimurium (GenBank accession number U61147)and E. coli (GenBank accession numbers X59593 and M60111). robA genesinclude those found in E. coli (GenBank accession numbers AE000509,U00096, M97495 and M94042).

[0034] Other known homologs of marA include those found inEnterobacteriaceac by nucleic acid hybridization under stringentconditions (Cohen et al., 1993). Other marA homologs include pqrA,identified in Proteus vulgaris (GenBank accession number D13561), ramAidentified in Klebsiella pneumonia (GenBank accession number U19581),and aarP identified in Providencia stuartii (GenBank accession numberL38718).

[0035] Additional homologs of marA (and other gene loci useful accordingto the invention) can be identified by conventional techniques. Suchtechniques include cloning by hybridization to marA or to known homologsthereof, and functional cloning. Cloning by hybridization involvessubjecting marA or known homologs thereof to hybridization with nucleicacids of bacteria (preferably the chromosomal DNA) under stringentconditions. The term “stringent conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references which compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. More specifically,stringent conditions, as used herein, refers, for example, tohybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mMNaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.15Msodium citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, the membrane uponwhich the DNA is transferred is washed at 2×SSC at room temperature andthen at 0.1×SSC/0.1×SDS at temperatures up to 65° C.

[0036] There are other conditions, reagents, and so forth which can beused, which result in a similar degree of stringency. The skilledartisan will be familiar with such conditions, and thus they are notgiven here. It will be understood, however, that the skilled artisanwill be able to manipulate the conditions in a manner to permit theclear identification of homologs and alleles of nucleic acids of theinvention. The skilled artisan also is familiar with the methodology forscreening cells and libraries for expression of such molecules whichthen are routinely isolated, followed by isolation of the pertinentnucleic acid molecule and sequencing.

[0037] In general homologs typically will share at least 30% nucleotideidentity and/or at least 40% amino acid identity to mar/sox/rob genes orto efflux pumps genes, or their polypeptide products respectively, insome instances will share at least 50% nucleotide identity and/or atleast 65% amino acid identity and in still other instances will share atleast 60% nucleotide identity and/or at least 75% amino acid identity.Watson-Crick complements of the foregoing nucleic acids also areembraced by the invention.

[0038] Functional cloning is useful to isolate homologs which do notshare sufficient homology at the nucleotide or amino acid sequence levelto permit cloning by nucleic acid hybridization, but which neverthelessare functional equivalents of the genes useful in the invention.Functional equivalents need not exhibit the same level of activity,merely activity of the same kind. For example, one phenotypicmanifestation of mara expression is the induction of the expression of aset of genes, including acrA. A gene which induces substantially thesame set of genes but at a lower level of expression would be considereda functional equivalent.

[0039] Functional cloning, as used herein, involves expression of anucleic acid sequence in a bacterium and determining whether theexpression of that sequence confers a desired phenotype on thebacterium. It is known that marA homologs exhibit similar functionalcharacteristics with respect to multiple antibiotic resistancephenotype. For example, overexpression of either soxS or robA in E. coliproduces both increased organic solvent tolerance and low-levelresistance to multiple antimicrobial agents (Ariza et al., J. Bacteriol.177:1665-1661, 1995; Nakajima et al., Biosci. Biotechnol. Biochem.59:1323-1325, 1995a; Nakajima et al., Appl. Environ. Microbiol.61:2302-2307, 1995b). Thus, for marA homologs, the desired phenotype canbe multiple antibiotic resistance, induction of mar-regulated genes(see, e.g., U.S. Pat. No. 5,650,321), and the like. For determiningmultiple antibiotic resistance, all that is necessary is to express theputative marA homolog in a non-multiple antibiotic resistant bacteriumand determine whether the modified bacterium acquires resistance to morethan one antibiotic, such as tetracycline, chloramphenicol, nalidixicacid, etc. marA homologs can be expressed according to standardprocedures, such as transformation with an expression plasmid containingthe marA homolog, introduction of one or more copies of the marA homologon the bacterial chromosome via transposon-mediated insertion, etc.

[0040] The acrAB locus, positively regulated by MarA (Ma et al., Mol.Microbiol. 16:45-55, 1995) and SoxS and RobA (Ma et al., Mol. Microbiol.19:101-112, 1996), specifies a proton-motive-force-dependent multidrugefflux pump for a wide variety of mostly lipophilic substances (Ma etal., 1995; Nikaido, Bacteriol. 178:5853-5859, 1996; Nikaido, Science264:382-387, 1994; Paulsen et al., Microbiol Rev. 60:575-608, 1996). Marmutants and wild type strains deleted of this locus become equallyhypersusceptible to antibiotics (Okusu et al., J. Bacteriol.178:306-308, 1996) suggesting that the acrAB pump confers an intrinsicresistance level which is then enhanced in Mar mutants.

[0041] The acrA and acrB genes have been cloned and sequenced. Forexample, the sequences of acrAB in E. coli are deposited as GenBankaccession number U00734. The acrAB genes have homologs in E. coli, aswell as in other species of bacteria. Sequence homologs of acrAB effluxpumps are referred to herein as “acr-like” efflux pumps. Isolation ofacr-like efflux pumps and other efflux pumps can be carried outaccording to the methods described above for nucleic acid hybridizationand functional cloning. Inhibitors of such acrAB homologs also areuseful for reducing the selection of antibiotic resistant bacterialmutants and potentiating the antibacterial properties of non-antibioticantibacterial compositions.

[0042] Agents which induce overexpression of acr-like efflux pumps areuseful in promoting organic solvent tolerance. Inducers of efflux pumpsinclude genes which encode the various efflux pumps which when expressedin a bacterium as a nucleic acid operably linked to a promoter canincrease the numbers of efflux pump protein molecules in the bacterium.Agents also include molecules which inhibit the function of efflux pumpregulatory genes. For example, antisense nucleic acids which bind toacrR and prevent its transcription or translation would function asinducers of acrAB. Efflux pumps can also be induced by mutation ofregulatory genes (such as acrR for the acrAB pump).

[0043] Agents useful in decreasing the expression or activity of anefflux pump for increasing organic solvent susceptibility (decreasingorganic solvent tolerance) are provided in the following paragraphs.

[0044] Agents which bind to a gene locus which mediates enhancedexpression of an efflux pump (such as the mar/sox/rob class of genes) ora nucleic acid expression product thereof include antisense nucleicacids, ribozymes and regulatory proteins such as repressor proteins(e.g. MarR). For example, antisense nucleic acids which bind to marA andprevent transcription or translation thereof would function asinhibitors of marA and agents which bind marA. Agents which bind to aprotein expression product of a gene locus include antibodies.Inhibitors of the foregoing gene loci and expression products alsoinclude molecules which bind to the gene loci and expression products asdescribed above. Other classes of agents and inhibitors of these typeswill be known to those of skill in the art.

[0045] Classes of inhibitors of efflux pumps useful in the methods andcompositions of the invention have been described previously in PCTpublished patent application WO96/33285 (includingL-phenylalanyl-L-arginyl-β-naphthylamide). Methods for testing compoundsfor efflux pump inhibition are also described therein. Other usefulinhibitors include ethanol (concentrations of about 4%), methanol,hexane and minocycline. Still other inhibitors include antisense nucleicacids and ribozymes directed against the gene(s) encoding the effluxpump. For example, antisense nucleic acids which bind to acrAB genes andprevent transcription or translation thereof would function asinhibitors of acrAB. Antibodies which bind efflux pumps or proteinswhich regulate the expression of efflux pumps are another class ofinhibitors. Still other inhibitors include genes which repressexpression of the efflux pumps or regulatory loci (such as marR) whichregulate expression of efflux pumps. Increasing the amount of such genesor the expression products thereof reduces the expression of effluxpumps in bacteria.

[0046] As mentioned above, the invention embraces antisense nucleicacids, including oligonucleotides, that selectively bind to a nucleicacid molecule encoding an efflux pump (e.g. acrA) or a molecule whichregulates expression of an efflux pump (e.g. marA). As used herein, theterm “antisense oligonucleotide” or “antisense” describes anoligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an RNA transcript of that geneand, thereby, inhibits the transcription of that gene and/or thetranslation of that RNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon the nucleic acid sequence of a gene of interest,one of skill in the art can easily choose and synthesize any of a numberof appropriate antisense molecules for use in accordance with thepresent invention. In order to be sufficiently selective and potent forinhibition, such antisense oligonucleotides should comprise at least 10and, more preferably, at least 15 consecutive bases which arecomplementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., NatureBiotechnol. 14:840-844, 1996). Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20-30 bases.Although oligonucleotides may be chosen which are antisense to anyregion of the gene or RNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted. Inaddition, the antisense is targeted, preferably, to sites in which RNAsecondary structure is not expected and at which proteins are notexpected to bind.

[0047] In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared bystandard methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

[0048] In preferred embodiments, however, the antisense oligonucleotidesof the invention also may include “modified” oligonucleotides. That is,the oligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

[0049] The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

[0050] The term “modified oligonucleotide” also encompassesoligonucleotides with a covalently modified base and/or sugar. Forexample, modified oligonucleotides include oligonucleotides havingbackbone sugars which are covalently attached to low molecular weightorganic groups other than a hydroxyl group at the 3′ position and otherthan a phosphate group at the 5′ position. Thus modifiedoligonucleotides may include a 2′-O-alkylated ribose group. In addition,modified oligonucleotides may include sugars such as arabinose insteadof ribose. The present invention, thus, contemplates preparationscontaining modified antisense molecules that are complementary to andhybridizable with, under physiological conditions, nucleic acidsencoding mar/sox/rob or efflux pump polypeptides, together with one ormore carriers.

[0051] As described above, the invention further embraces the use ofantibodies or fragments of antibodies having the ability to selectivelybind to efflux pumps, as well as polypeptides which regulate theexpression of efflux pumps. Antibodies include polyclonal and monoclonalantibodies, prepared according to conventional methodology.

[0052] Significantly, as is well-known in the art, only a small portionof an antibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation. Any of the foregoing antigen fragments are useful in themethods and compositions of the invention. The present invention alsoincludes so-called single chain antibodies and intracellular antibodies.

EXAMPLES Example 1

[0053] Mutants resistant to Pine-Sol/pine oil were obtained fromstationary phase LB broth cultures of E. coli strain “WEC” (wild typestrain 15-5068 from Carolina Biological Supply Co., Burlington, N.C.)and AG100 (George and Levy, J. Bacteriol. 155:531-540, 1983), at 30° C.on nutrient agar (NP 3.5 GP) or LB agar with 2-3 days incubation in avariety of ways: using a 6 mm disc method; plating cells on plates orgradient plates (Curiale and Levy, J. Bacteriol. 151:209-215, 1982),containing PINE-SOL™ (product of Clorox Co., Oakland, Calif.) or pineoil itself.

[0054] Antibiotic susceptibility was measured at 30° C. using antibioticsusceptibility discs (Carolina Biological), gradient plates with thedrug in the top agar (Curiale and Levy, 1982) or agar dilution plates(concentration steps of 1.5 fold; inocula of 10⁵ cells/5 μl spot). Whilethere was a variability of resistance phenotypes, all Pine-Sol/pineoil-selected mutants were also multidrug resistant (Table 1 A, B). TABLE1A Susceptibility by discs; diameter of clearing (mm) StrainCharacteristics Ap Cm Tc WEC wild type 22 27 21 NP3.5GP mutant of WECselected on Pine- 12 11 14 Sol gradient (0-1.5%)

[0055] TABLE 1B Susceptibility by gradient plates (MIC)^(b) (% byvolume) (μg/ml) Strain Characteristics PS Ap Cm Nal Tc AG100 wild type0.9 <1.2 2.6 1.7 1.8 AP1 mutant of AG100 >3.6/0.9 3.0 7.8 9.7 2.4selected by pine oil on disc AP5 mutant of AG100 >2.9/0.9 7.2 21 7.5 4.5selected as for AP1 APS3 mutant of AG100 1.8 7.7 >35 8.6 5.3 selected onPine- Sol gradient (0-1.5%) AG102 [ 1 ]^(d) Mar mutant of >4.1 8.5 >3514.0 >12.8 AG100, selected on Tc (2 steps) HH180 [ 2 ]^(d) deletion of39 kb 0.3 <0.6 ND^(c) <1.8 <0.6 including mar locus; has zdd- 230::Tn9(Cm^(R)); in host strain MM294 HH188 [ 2 ]^(d) HH180 containing 0.9 <1.0ND^(c) 3.7 1.2 pHHM183 (mar+) HH191 [ 2 ]^(d) HH180 containing 2.3 5.4ND^(c) 9.1 8.2 pHHM191 (marR2) HH193 [ 2 ]^(d) HH180 containing 3.2 5.9ND^(c) 10.9 >11.4 pHHM193 (marR5) [ 3 ]^(d)

[0056] In host strain HH180, deleted of the entire mar region, plasmidpHHM188, bearing a 9 kb wild type mar caused no change in the resistancephenotype. In the same host pHHM191 and pHHM193 each containing cloned 9kb fragment including the entire mar locus, and marR was mutant, causeda Mar phenotype (Cohen et al., J. Bacteriol. 175:1484-1492, 1993). TheseMar mutants, as well as AG102 were resistant to Pine-Sol (Table 1B) andto 100% pine oil when compared to their respective wild type strains.TABLE 2 Effect of inactivation of mar, sox, rob, or acr locus uponsusceptibility to Pine-Sol Relative MIC for Pine-Sol^(a) Strain mar^(b)sox^(b) rob^(b) acr^(b) AG100 1 0.9 0.8 <0.06 AP1 0.5 <0.6 0.5 <0.02 AP50.4 0.9 0.8 <0.02 APS3 0.4 1 1 <0.04 AG102 0.4 1 1 <0.03

[0057] Northern blot analysis for expression of marA mRNA using a[radiolabeled] marA probe in the absence and presence of the inducersalicylate (Cohen et al., J. Bacteriol. 175:7856-7862, 1993) revealedthat, like Mar mutant AG102, mutants AP5 and NP3.5 GP showed an overexpression of marA that was enhanced by salicylate (FIG. 1). Overexpression was also seen in mutant APS3 (data not shown). The wild typeAG100 and the pine oil mutant AP1 showed no detectable signal (FIG. 1).We concluded that AP5, NP3.5GP, and APS3, but not AP1, were probably Marmutants.

[0058] The marCORAB locus was deleted in the Pine-Sol/pine oil mutantsand in AG102 by P1 transduction (Provence and Curtiss, p. 317-347, InGerhardt et al., eds., Methods for General and Molecular Bacteriology.ASM, Washington, D.C., 1994) using AG100/Kan (Maneewannakul and Levy,1996) as the donor strain and selecting on kanamycin. The deletioncaused a 60-70% reduction in the resistance of mutants to Pine-Sol(Table 2), down to approximately a wild type level. The same was truefor mutant NP3.5GP (data not shown).

[0059] Inactivation of the soxRS and robA loci in the Pine-Sol/pine oiland Mar mutants via P1 transduction of a kanR gene in the gene causeddecreased resistance to Pine-Sol only in the mutant AP1 (Table 2).Mutant AP1 did not overexpress mar or soxRS (data not shown). Deletionof the acrAB locus by P1 transduction of kanR in the gene increasedsusceptibility to Pine-Sol in all strains (Table 2).

[0060] Deletion of acrAB (but not of mar) also caused more than a tenfold increase in the susceptibility of strains to the productscontaining the quaternary amine or chloroxylenol (data not shown),suggesting that AcrAB was also involved in effluxing those twodisinfectants.

Example 2

[0061] Organic solvent tolerance mediated by the marA, soxS, robA andacrAB loci of E. coli is described in White et al. (J. Bacteriol.179:6122-6126, 1997). These results are summarized below. AG102, a Marmutant of AG100, grew in the presence of n-hexane, cyclohexane (Table3), and n-pentane (data not shown) whereas AG100 grew only in hexane.TABLE 3 Organic solvent tolerancc of wild-type and mar strains bearingmar, soxS, or robA plasmids. Growth in presence of organic solvent^(a)Strain Plasmid^(b) n-hexane (3.9)^(c) cyclohexane (3.4) AG102 (marR ++++ mutation) AG100 (wild-type) ++ − AG100 pMAK-TU1 ++ − AG100 pMAK-TU2++ + AG100 pMAK-TU1 & ++ ++ TU2 AG100 pSMarAB ++ + AG100 pSXS ++ ++AG100 pSRob ++ ++ AG100K + − (marCORAB::kan) AG100K pMAK-TU1 + − AG100KpMAK-TU2 ++ + AG100K pMAK-TU1 & ++ ++ TU2 AG100K pSMarAB ++ + AG100KpSXS ++ ++ AG100K pSRob ++ ++ MCH164 (Δmar) + − MCH164 pMAK-TU1 + −MCH164 pMAK-TU2 ++ − MCH164 pMAK-TU1 & ++ ++ TU2 MCH164 pSMarAB ++ −MCH164 pSXS ++ − MCH164 pSRob ++ − AG100-B (acrR ++ + mutant) AG100-A(ΔacrAB) − − AG102-A (marR1, − − ΔacrAB) AG102-A pSMarAB − − AG102-ApSXS − − AG102-A pSRob − −

[0062] In the wild type E. coli AG100 background, over expression ofmarA (on plasmid pSMarAB or pMAK-TU2) or soxS (on pSXS) or robA (onpSRob) resulted in cyclohexane tolerance (Table 3). marC by itself(pMAK-TU1) had no effect on cyclohexane tolerance, however, introductionof marCORAB on the low copy plasmid pMAK705 (pMAK-TU1 & TU2) resulted incyclohexane tolerance (Table 3).

[0063] When the mar locus was inactivated by replacement with akanamycin resistance cassette (AG100K) (Maneewannakul and Levy, 1996),the strain became hypersusceptible to n-hexane as compared to the wildtype strain (Table 3). MCH164 [a derivative of AG100 from which 39 kb ofchromosomal DNA including the entire mar locus had been deleted (Goldmanet al., Antimicrob. Agents Chemother. 40:1266-1269, 1996; McMurry etal., Antimicrob. Agents Chemother. 38:542-546, 1994)] was, as expected,also hypersusceptible to organic solvents (Table 3). Expression in transof marA, soxS, or robA in AG100K, restored n-hexane tolerance, andincreased cyclohexane tolerance in the cell (Table 3). Expression intrans in AG100K of marA, specified from plasmid pMAK-TU1 & TU2 restoredn-hexane tolerance and produced higher cyclohexane tolerance (Table 3).While introduction of either marA, soxS, or robA restored n-hexanetolerance in MCH164, only pMAK-TU1 TU2 produced cyclohexane tolerance inthis larger deletion mutant (Table 3).

[0064] Overexpression of acrAB, because of a mutation in acrR inAG100-B, enabled the strain to grow in the presence of cyclohexane(Table 3). Deletion of acrAB from wild-type AG100 (AG100-A) resulted inn-hexane sensitivity (Table 3). Deletion of acrAB from the Mar mutant(AG102-A) resulted in both n-hexane and cyclohexane sensitivity.Expression of marA, soxS, or robA in AG102-A failed to restore organicsolvent tolerance, further demonstrating the critical role of acrAB(Table 3).

[0065]E. coli strains overexpressing MarA (JHC1069; cfxB1/MarR mutation)or SoxS (JTG1078; soxR105 mutation) grew in the presence of bothn-hexane and cyclohexane, whereas the wild-type C4468 only grew in thepresence of n-hexane (Table 4). Much like the situation in AG100,introduction of either pSMarAB, pMAK-TU2, pMAK-TU1 & TU2, pSXS, or pSRobinto GC4468 produced cyclohexane tolerance. Inactivation of robA byinsertion of a kanamycin cassette (RA4468) caused n-hexanesusceptibility (Table 4). Introduction of either marA (on pMAK-TU1 &TU2, pMAK-TU2, or pSMarAB), SoxS (on pSXS), or RobA (on pSRob), into therobA inactivated strain, increased both n-hexane and cyclohexanetolerance (Table 4). Deletion of soxRS (DJ901) had little effect onn-hexane tolerance (Table 4). Introduction of marA, soxS, or robA intothe ΔsoxRS strain produced cyclohexane tolerance (Table 4). In all thesecomplementations, the effect of marA was best noted from plasmidpMAK-TU1 & TU2. TABLE 4 Organic solvent tolerance of wild-type, ΔsoxRS,or robA::Kan strains bearing mar, soxS, or robA plasmids. Growth inpresence of organic solvent^(a) Strain Plasmid^(b) n-hexane (3.9)^(c)cyclohexane (3.4) GC4468 (wild-type) ++ − JHC1069 (cfxB1) ++ ++ JTG1078(soxR105) ++ ++ GC4468 pMAK-TU1 ++ − GC4468 pMAK-TU2 ++ + GC4468pMAK-TU1 & ++ ++ TU2 GC4468 pSMarAB ++ + GC4468 pSXS ++ ++ GC4468 pSRob++ ++ RA4468 (robA::kan) + − RA4468 pMAK-TU1 + − RA4468 pMAK-TU2 ++ +RA4468 pMAK-TU1 & ++ ++ TU2 RA4468 pSMarAB ++ + RA4468 pSXS ++ ++ RA4468pSRob ++ ++ DJ901 (AsoxRS) ++ − DJ901 pMAK-TU1 ++ − DJ901 pMAK-TU2 ++ +DJ901 pMAK-TU1 & ++ ++ TU2 DJ901 pSMarAB ++ + DJ901 pSXS ++ ++ DJ901pSRob ++ ++

[0066] These results show that overexpression of marA, soxS, or robAleads to increased organic solvent tolerance and that tolerance ismediated by the acrAB efflux pump.

[0067] Equivalents

[0068] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

[0069] All references disclosed herein are incorporated by reference.

We claim:
 1. A method for inhibiting the selection or propagation of abacterial mutant that overexpresses an efflux pump comprising:contacting bacteria with an agent that binds to a gene locus or anexpression product thereof, wherein the expression of the gene locusenhances expression of the efflux pump, in an amount effective toinhibit the gene locus-enhanced expression of the efflux pump.
 2. Themethod of claim 1, wherein the gene locus is selected from the groupconsisting of a mar locus, a sox locus and a rob locus.
 3. The method ofclaim 2, wherein the gene locus is marA.
 4. The method of claim 2,wherein the gene locus is soxS.
 5. The method of claim 2, wherein thegene locus is robA.
 6. The method of claim 1, wherein the efflux pump isacr-like.
 7. The method of claim 6, wherein the efflux pump is acrAB. 8.The method of claim 1, wherein the agent is selected from the groupconsisting of antisense nucleic acids, antibodies, ribozymes, chemicalsand proteins which repress expression of the gene locus.
 9. The methodof any of claims 1-8, wherein the agent is an antisense nucleic acids.10. A method for rendering bacterial cells more susceptible to anon-antibiotic bactericidal or bacteriostatic agent that is a substrateof an efflux pump comprising: administering to the bacterial cell aninhibitor of a gene locus or an expression product thereof, wherein theexpression of the gene locus enhances expression of an efflux pump. 11.The method of claim 10, wherein the gene locus is selected from thegroup consisting of a mar locus, a sox locus and a rob locus.
 12. Themethod of claim 11, wherein the gene locus is marA.
 13. The method ofclaim 11, wherein the gene locus is soxS.
 14. The method of claim 11,wherein the gene locus is robA.
 15. The method of claim 10, wherein theefflux pump is acr-like.
 16. The method of claim 15, wherein the effluxpump is acrAB.
 17. The method of claim 10, wherein the inhibitor isselected from the group consisting of antisense nucleic acids,antibodies, ribozymes, chemicals and proteins which repress expressionof the gene locus.
 18. The method of any of claims 10-17, wherein theinhibitor is an antisense nucleic acid.
 19. A method for renderingbacterial cells more susceptible to a non-antibiotic bactericidal orbacteriostatic agent that is a substrate of an efflux pump comprising:administering to the bacterial cell an inhibitor of the efflux pump. 20.The method of claim 19, wherein the efflux pump is acr-like.
 21. Themethod of claim 20, wherein the efflux pump is acrAB.
 22. The method ofclaim 19, wherein the inhibitor is selected from the group consisting ofL-phenylalanyl-L-arginyl-β-naphthylamide, 4% ethanol, methanol, hexane,minocycline.
 23. The method of any of claims 19-22, wherein theinhibitor is L-phenylalanyl-L-arginyl-β-naphthylamide.
 24. A method forincreasing the ability of bacterial cells to survive in an organicsolvent comprising: enhancing expression in the bacterial cells of anorganic solvent bacterial efflux pump by growing the bacterial cells inthe presence of a non-mar/sox/rob agent that induces the overexpressionof the organic solvent bacterial efflux pump.
 25. The method of claim24, wherein the agent is a gene encoding an acr-like pump or anexpression product thereof.
 26. The method of claim 25, wherein theacr-like pump is acrAB.
 27. The method of claim 24, wherein the agent isselected from the group consisting of an antibiotic, and anon-antibiotic antibacterial compound.
 28. A method for decreasing theability of bacterial cells to survive in an organic solvent comprising:reducing expression in the bacterial cells of an organic solventbacterial efflux pump by growing the bacterial cells in the presence ofan agent that reduces the expression of the organic solvent bacterialefflux pump.
 29. The method of claims 28, wherein the agent is aninhibitor of a gene locus or an expression product thereof, wherein theexpression of the gene locus enhances expression of an efflux pump. 30.The method of claim 29 wherein the gene locus is selected from the groupconsisting of a mar locus, a sox locus and a rob locus.
 31. The methodof claim 30 wherein the gene locus is marA.
 32. The method of claim 30wherein the gene locus is soxS.
 33. The method of claim 30 wherein thegene locus is robA.
 34. The method of claim 29 wherein the efflux pumpis acr-like.
 35. The method of claim 34 wherein the efflux pump isacrAB.
 36. The method of claim 29 wherein the inhibitor is selected fromthe group consisting of antisense nucleic acids, antibodies, ribozymes,chemicals and proteins which repress expression of the gene locus. 37.The method of any of claims 28-36 wherein the inhibitor is an antisensenucleic acid.
 38. A method for testing the ability of a non-antibioticcomposition to induce a multiple antibiotic resistance phenotype in abacterium comprising (a) contacting the bacterium with thenon-antibiotic composition, (b) determining the expression of abacterial gene locus, the altered expression of which is indicative ofinduction of the multiple antibiotic resistance phenotype in thebacterium, and (c) comparing the result of (b) with a control, whereinaltered expression of the bacterial gene locus indicates that thenon-antibiotic composition induces the multiple antibiotic resistancephenotype in the bacterium.
 39. The method of claim 38, wherein the genelocus is selected from the group consisting of a mar locus, a sox locus,a rob locus and an acr-like efflux pump locus.
 40. The method of claim39, wherein the gene locus is marA.
 41. The method of claim 39, whereinthe gene locus is soxS.
 42. The method of claim 39, wherein the genelocus is robA.
 43. The method of claim 39, wherein the efflux pump isacr-like.
 44. The method of claim 43, wherein the efflux pump is acrAB.45. The method of 38, wherein the composition is an inactive ingredient.46. The method of claim 45, wherein the inactive ingredient is anon-bactericidal ingredient.
 47. The method of claim 45, wherein theinactive ingredient is a non-bacteriostatic ingredient.
 48. The methodof claim 38, wherein step (b) is performed by determining the enzymaticactivity of an expression product of a marker gene fused to thebacterial gene locus.
 49. The method of claim 48, wherein the markergene is lacZ.
 50. A composition comprising: a non-antibioticbactericidal or bacteriostatic first agent and a second agent thatinhibits the expression of or activity of an efflux pump.
 51. Thecomposition of claim 50, wherein the second agent inhibits theexpression of a gene locus or an expression product thereof, wherein theexpression of the gene locus enhances expression of the efflux pump. 52.The composition of claim 51, wherein the second agent is selected fromthe group consisting of antisense nucleic acids, antibodies, ribozymes,chemicals and proteins which repress expression of the gene locus. 53.The composition of claim 52, wherein the second agent is an antisensenucleic acid.
 54. The composition of claim 50, wherein the second agentinhibits an acr-like efflux pump.
 55. The composition of claim 54,wherein the second agent is selected from the group consisting ofL-phenylalanyl-L-arginyl-β-naphthylamide, 4% ethanol, methanol, hexane,minocycline.
 56. The method of claim 55, wherein the second agent isL-phenylalanyl-L-arginyl-β-naphthylamide.
 57. The composition of claim50, wherein the first agent is selected from the group consisting oftriclosan, pine oil, quaternary amine compounds including alkyl dimethylbenzyl ammonium chloride, chloroxylenol, triclocarbon, disinfectants andorganic solvents.
 58. A method for identifying an antibacterialcomposition which does not select or induce a multiple antibioticresistance phenotype in a bacterium, comprising (a) contacting thebacterium with the antibacterial composition, (b) determining theexpression of a bacterial gene locus, the altered expression of which isindicative of induction of the multiple antibiotic resistance phenotypein the bacterium, and (c) comparing the result of (b) with a control,wherein a lack of altered expression of the bacterial gene locusindicates that the antibacterial composition induces the multipleantibiotic resistance phenotype in the bacterium.
 59. The method ofclaim 58, wherein the gene locus is selected from the group consistingof a mar locus, a sox locus, a rob locus and an acr-like efflux pumplocus.
 60. The method of claim 59, wherein the gene locus is marA. 61.The method of claim 59, wherein the gene locus is soxS.
 62. The methodof claim 59, wherein the gene locus is robA.
 63. The method of claim 59,wherein the efflux pump is acr-like.
 64. The method of claim 63, whereinthe efflux pump is acrAB.
 65. The method of claim 58, wherein step (b)is performed by determining the enzymatic activity of an expressionproduct of a marker gene fused to the bacterial gene locus.
 66. Themethod of claim 65, wherein the marker gene is lacZ.